Microtubles are critically involved in a variety of cellular processes, including mitosis. They are composed primarily of the protein tubulin. The research described here will apply high resolution biochemical techniques to the question of how microtubule assembly is regulated in vitro and in vivo, focusing largely on the role of tubulin's sulfhydryl groups. A model system has been constructed in which (14C)iodoacetamide is used to label the sulfhydryls and inhibit assembly in vitro. This system will be applied to tubulin prepared by different methods and, for each kink of tubulin, the following information will be elicited; the titers of free sulfhydryls, intra-chain disulfides, and protein-bound glutatione; the number and location (in the primary and tertiary structures) of the sulfhydryls critical in regulating assembly is inhibited; and the conformational effects of alkylation. The results should exactly define the molecular mechanims which regulates microtubule assembly. The method will be applied to tubulin isolated from synchronized cultured cells to test and amplify the hypothesis that the status of tubulin's sulfhydryl groups is an important factor in the regulation of microtubule assembly during the cell cycle. The role of tubulin phosphorylation will also be investigated by an approach whereby the phosphate group will be removed enzymatically and the assembly and conformational properties of the de-phosphorylated tubulin will be examined. Finally, the existence and nature of oligosaccharides covalently bound to tubulin will be determined using large quantities of highly pure alpha- and beta-tubulin from different axonemal fractions from sea urchin sperm.